Inr J Raduxm Oncdogy Btol. Phys.. Vol. 10. PP. Iu)I-1305 0X0-3016/84 503.00 t .JO Printed in the U.S.A. All nghe mcmd. Copyr&Jtl Q 1984 F-erwnon Press Ltd.

??Session II

PHARMACOKINETIC STUDIES OF AMINO ACID ANALOGUES OF 2-NITROIMIDAZOLE, NEW HYPOXIC CELL RADIOSENSITIZERS

KRISHNA C. AGRAWAL, PH.D., CYNTHIA A. LARROQUETTE, B.S. AND PRADEEP K. GARG, PH.D.

Department of Pharmacology, Tulane University School of Medicine, New Orleans, LA 70112

A series of new analogues of 2-nitroimidazoie has been synthesixed by inserting various amino acids at l-position through an amide bond. The ethyl esters of N-a_1(2ritro-l-imidazoiyi)acetyi)-L-phenylaIanine and N-o-(2-nit- I-imidazolyi)acetylj-Ltyrosine were found to be the most effective radiosensitizers in vitro against hypoxic Chii hamster (V-79) cells. The sensitizer enhancement ratios (SER) of these derivatives were 2.2 and 2.3 respectively at 1 mM concentration after 2 hr exposure under hypoxia. However, the free acid of phenylalanine nnaiogue was less active as a radiosensitizer and required 5 mM concentration to produce SER of 1.9. In contrast, the free acid of tyrosine analogue was inactive in this test system. The pharmacokinetic studies with the esters revealed their rapid hydrolysis in serum to the corresponding acids within 5 minutes as detected by HPLC. The pharmacokinetic parameters were therefore determined by employing the free acid anaiogues and soiubiliing them as their sodium salts. The drugs were administered intraperitoneally at 0.5 me/g dose level to C-57 mice bearing B16 melanoma. These agents were cleared from the plasma rapidly with an apparent tllr of 188 and 15.6 min respectively. Peak tumor concentration of approxhuateiy 217 ug/g was achieved within 15 min with phenylalanine anaiogue. The tumor to brain ratio was 1O:l suggesting that this agent is excluded from CNS and that the phenylalanine analogue should be considered a potentially less neurotoxic radiosensitizer than misonidazoie.

Amino acid analogues, 2-Nitroimidaaoies, RadiosensitizPtion, Chinese hamster ceils, Pharmacokinetics.

INTRODUCI’ION

During the past few years, a variety of structural analogues of misonidazole has been synthesized in an effort to obtain a therapeutically superior radiosensitizer of hypoxic tumor cells.’ The major problem associated with the clinical trials of misonidazole has been the dose related neuro- toxicity.’ To alleviate this limitation, efforts have been made to develop agents with lower partition coefhcients than misonidazole.’ One of these analogues, SR-2508, is currently under clinical investigation.

In our systematic approach of design and synthesis of 2nitroimidazole analogues, we have repotted synthesis and biological activity of a series of 2-nitroimidazole nu- cleosides.4 It was envisioned that since nuclecsides em- ployed in cancer chemotherapy do not cross the blood brain barrier in effective concentration, this class of an- alogues may provide agents with increased therapeutic efficacy. Indeed, one of these agents, I-(2’,3’-dideoxy-cY- D-er_vfhro-hex-2’-enopyranosyl)-2nitroimidazole (RA- 263) was found to be superior to misonidazole as a ra- diosensitizer.4 Another new approach has now been ex- plored in our laboratory in which the amino acid transport

mechanism and the endogenous membrane bound amino acid transferases and peptidases in solid tumors are uti- lized. Accordingly, we have synthesized a series of amino acid analogues of 2-nitroimidazole by inserting various amino acids through an amide bond at l-position of the 2nitroimidazole moiety.3 It was anticipated that the new amino acid derivatives would be actively transported and then hydrolyzed in solid tumors to release the 2-nitroim- idazole moiety that would then passively diffuse into the hypoxic cells. These analogues were also expected to be excluded from the CNS because of increased hydrophil- icity. In this report we have investigated the in vitro ra- diosensitization and pharmacokinetics of the phenylal- anine and tyrosine analogues (Fig. 1).

METHODS AND MATERIALS

Compounds The compounds used in this investigation are shown

in Fig. 1 and were synthesized in our laboratory. The methods of synthesis and physicochemical parameters of these new agents will be submitted for publication else- where.

This investigation was supported by PHS Grant Number CA- 21050. awarded by the National Cancer Institute. DHHS.

Reprint requests to: Krishna C. Agrawal, Ph.D. Accepted for publication ,22 March 1984.

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1302 Radiatton Oncology ??Biology 0 Physics August 1984. Volume IO. Number 8

iOOR'

I, R = H, R' = C2H5

II, R = OH, R' = C2H5

III, R = H, R' = H

Iv, R = OH, R' = H Fig. I. Structures of amino acid analogues of 2-nitroimidazole.

Cell culture studies The cytotoxicity and radiosensitization studies were

carried out by employing asynchronous monolayer cul- tures of Chinese hamster (V-79) cells. The techniques employed for these experiments have been reported ear- lier.’ The test compounds I and II were water insoluble and were therefore dissolved in 1% DMSO in minimal essential medium (MEM) and diluted with MEM to ap- propriate concentration. Compounds III and IV were wa- ter soluble and were directly dissolved in MEM. The pH of MEM was 7.4 except in experiments where the effect of lowering the pH on biological activity was investigated. The cytotoxicity experiments were carried out at 37°C under oxic and hypoxic conditions. Hypoxia was achieved by purging the sealed containers with 95% N2/5% CO*. Cells were irradiated at room temperature with a linear accelerator at a dose rate of 180 rad/min.

Pharmacokinetic studies The pharmacokinetic studies were carried out in 07

mice bearing B I6 melanoma, two weeks after tumor im- plantation when the tumors were approximately I50 mm3 in size. The tumors were transplanted in the right hind leg subcutaneously with lo6 cells in 0.1 ml saline. The cells were obtained from a tumor of a donor mouse. The amino acid analogues were dissolved by addition of 1 N sodium bicarbonate solution and administered intraper- itoneally at a dose level of 0.5 mg/g (1.57 mmoles for compound III and 1.50 mmoles for compound IV). Plasma and tissue homogenates were extracted with

methanol and analyzed by reversed phase (Clx Bio-sil ODS- 10 column) high-performance liquid chromatog- raphy (Beckman 165 detector) employing 40% methanol. 60% water and 0.5% acetic acid as mobile phase. The peak was detected at 320 nm with a retention time of 6.8 and 3.1 min for compounds III and IV respectively. The flow rate was 1 ml/min.

RESULTS

Initially, the radiation survival curves were obtained with the ethyl esters I and II at 1 mM against V-79 cells as shown in Fig. 2. The survival characteristics of these cells were similar to those exposed with misonidazole (1 mM), suggesting an analogous mechanism. Both I and II were significantly more effective sensitizers than mi- sonidazole at an equimolar concentration. The sensiti- zation was concentration dependent as has been observed with most misonidazole analogues. The enhancement ra- tios (ER) for compounds I and II were 2.2 and 2.3, re- spectively, in comparison to 1.9 for misonidazole (Ta- ble 1).

The acids III and IV were distinctly less effective as sensitizers of V-79 cells in vitro (Table 1). The tyrosine analogue IV was inactive as a sensitizer up to a concen- tration of 5 mM whereas the phenylalanine derivative III produced an ER of 1.4 at 2 mM and 1.9 at 5 mM con- centration. Experiments were also conducted in medium of lower pH of 6.0, 6.4, 6.8 and 7.0 in an attempt to increase the cellular uptake of free acids. However, no significant increase either in cytotoxicity or radiosensi- tization was observed. Compounds I (Fig. 3A) and II (Fig. 3B) were essentially nontoxic to V-79 cells up to a con- centration of 2 mM upon either 2 or 5 hr exposure. However, under hypoxic conditions, both these agents were differentially more toxic to V-79 cells and caused >60% inhibition of colony formation upon 2 hr exposure at 2 mM concentration. Hypoxic cytotoxicity was sig- nificantly increased as a function of time and by 5 hr resulted in almost complete inhibition of colony for- mation.

The pharmacokinetic experiments were initially con- ducted with the esters I and II; however, HPLC analysis indicated rapid and complete hydrolysis of the esters to corresponding acids. Therefore, the pharmacokinetic pa- rameters were determined with the acids III and IV in C57 mice bearing B 16 melanoma. The peak plasma levels, after intraperitoneal injection of either compound III or IV at a dose level of 0.5 mg/g were achieved within 15 min at the level of 1082 and 1333 ug/ml (Fig. 4), re- spectively. By 2 hr, these agents were essentially eliminated from plasma. The apparent plasma half-life (t,,r) was 18.8 and 15.6 min respectively. The concentration of com- pounds III and IV in tumor paralleled the plasma con- centration achieving the peak tumor concentration at 15 min at the level of 2 17 ug/g and 110 ug/g respectively.

Amino acid analogues of 2-nitroimidazole ??K. C. AGRAWAL er a/ 1303

1.0

0,001 0 1000 2000 3000

MDIATiON DOSE (bD)

Fig. 2. Radiation survival curves against hypoxic Chinese hamster cells: X, 1 mM misonidazole, A, I mM phenylalanine analogue (I), and 0, 1 mM tyrosine analogue (II).

The concentration of these agents in brain was also highest at 15 min at the level of 19.5 and 8.1 ug/g respectively.

The acute LDso (24 hr) of compounds III and IV were

Table I. In vifro radiosensitization of hypoxic Chinese hamster (V-79) cells by amino acid analogues of 2-nitroimidazole

Compound PC*

Concen- tration (mM)

Survival (W1

Enhance- ment ratio

(ER)

I 33.4 1.0 86.6 2.2 II 5.72 1.0 99.2 2.3

III 0.08 1.0 100.0 1.0 2.0 99.8 1.4 5.0 99.5 1.9

IV 0.01 1.0 97.8 1.0 2.0 98.6 1.0 5.0 95.5 1.0

Misonidazole 0.43 1.0 98.0 1.9

* Partition coefficients were determined in octanollphosphate buffer pH 7.4 at room temperature.

determined in C57 mice and were found to be >6.0 g/kg indicating that the amino acid analogues are greater than four-fold less toxic than misonidazole. The partition coefficients (PC) of these agents were determined in oc- tanol/phosphate buffer (pH 7.4) and are shown in Table 1. The esters I and II were highly lipid soluble with PC of 33.4 and 5.72 whereas the acids III and IV were hy- drophilic with PC of 0.08 and 0.01 respectively.

DISCUSSION

The development of various analogues of misonidazole has been directed primarily either to increase the electron affinity (for increased potency) or decrease the partition coefficient (for decreased neurotoxicity).’ In our system- atic approach we have initially reported synthesis of a series of nucleosides in an attempt to both increase the. cellular uptake and decrease the CNS toxicity because of lower PC of nucleosides.4 We have recently reported syn- thesis of a series of amino acid analogues in order to

Radiation Oncology 0 Biology 0 Physics August 1984. Volume 10, Number 8

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_ _

0.5 1.0 2.0

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Fig. 3. Hypoxic cytotoxicity against Chinese hamster cells: [A] effect of phenylalanine analogues (I), [B] effect of tyrosine an- alogue (II); X - X, 2 hr oxic; X - - - X, 2 hr hypoxic; A - A, 5 hr oxic; A - - - A, 5 hr hypoxic.

utilize the amino acid transport mechanism.2 In this re- port, we have described the in vitro radiosensitization and pharmacokinetic parameters of phenylalanine and ty- rosine analogues.

The in vitro radiosensitization studies have demon- strated that both the esters I and II are significantly more effective radiosensitizers than misonidazole at equimolar concentration achieving an ER of 2.2 and 2.3 respectively. However, the esters I and II are rapidly hydrolyzed in

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Fig. 4. Pharmacokinetics of compounds III and IV in C57 mice bearing B16 melanoma: [A], plasma concentration q/ml: A - A, III; X - X, IV: [B], tumor concentration ug/g; X - X, III; A - A, IV; brain concentration ug/g: X - - - X, III; A - - - A, IV.

vivo by esterases to the corresponding acids III and IV. The in vifro experiments with the acids indicated that III with PC of 0.08 was an active radiosensitizer; however,

Amino acid analogua of 2-nitroimidazole 0 K. C. AGRAWAL n al. 1305

it required a concentration of 5 mM to achieve a same degree of sensitization as 1 mM misonidazole (Table 1). The tyrosine analogue IV with PC of 0.01 was inactive, suggesting that perhaps the free acid was either completely ionized at pH 7.4 or was too hydrophilic to achieve ad- equate intracellular concentration. The experiments were therefore conducted at pH of lower than 7.4 to minimize ionization of the acids; however, no significant increase in biological activity was obtained. The pharmacokinetic experiments have demonstrated that the compound IV with PC of 0.01 was able to reach to the tumor tissue suggesting an uptake role of amino acid active transport mechanism. It is conceivable, however, that the concen- tration in tumor tissue may be extracellular.

The in vitro cytotoxicity experiments have shown that the esters I and II are significantly more cytotoxic to hypoxic cells than oxic cells even at short exposure time of 2 hr. By 5 hr, these agents completely inhibited the colony formation. It is not obvious whether this increased hypoxic cytotoxicity involves increased drug uptake under hypoxia or increased anaerobic metabolism to toxic spe-

cies.6 The high lipid solubility of the esters could lead to increased cellular uptake.

The pharmacokinetic experiments indicate that the tu- mor concentration (2 17 ug/g) achieved with compound III at 15 min after intraperitoneal injection of 1.57 mmoles of the drug may not be sufficient for radiosensitization. However, the dosage of compound III can be safely in- creased 5 to 10 fold because of its low toxicity. This would presumably provide an adequate concentration of the drug in tumor for radiosensitization. Although plasma, tumor and brain levels seem to be parallel to each other (Fig. 4), there is greater than lo-fold difference between tumor and brain concentration. Moreover, by 2 hr the drug is completely eliminated from the brain tissue in- dicating that the compound III may be less neurotoxic than misonidazole. The short apparent plasma t1,2 of 18.8 min also suggest that the drug is rapidly eliminated. Since the maximum degree of radiosensitization can be achieved at the peak levels of the compound in tumor, a reduced area under the curve (AUC) because of rapid elimination will be of increased therapeutic value.

REFERENCES

Adams, G.E., Ahmed, I., Clarke, E.D., CYNeill, P., Parrick, J., Stratford, I.J., Wallace, R.G., Wardman, P., Watts, M.E.: Structure-activity relationships in the development of hyp oxic cell radiosensitizers. III. Effects of basic substituents in nitroimidazole sidechains. Int. J. Radiat. Biol. 38: 6 I3- 626, 1980. Agrawal, K.C., Larroquette, L.A., Gupta, R.P.: New amino acid derivatives as carriers of 2-nitroimidazole moiety for radiosensitization (Abstr.). Radial. Rex 94: 611, 1983. Agrawal, KC., Miller, B.C., Neta, P.: Radiosensitization of hypoxic mammalian cells by dinitroimidazoles. Radial. Res. 78: 532-541, 1978. Agrawal, K.C., Sakaguchi, M., Rockwell, S.: A new potent radiosensitizer: 1-( 2’,3’-dideoxy-a-D-erythro-hex-2’-eno-

5.

6.

7.

pyranosyl)-2-nitroimidazole (RA-263). Int. J. Radiation Oncol. Biol. Phys. 8: 403-401, 1982. Brown, J.M., Workman, P.: Partition coefficient as a guide to the development of radiosensitizers which are less toxic than misonidazole. Radial. Res. 82: I7 l-190, 1980. Middlestadt, M.V., Rauth, A.M.: The effect of reduction products of misonidazole on Chinese hamster ovary cells. Int. J. Radiation Oncol. Biol. Phys. 8: 709-712, 1982. Urtasun, R.C., Chapman, J.D., Feldstein, M.L., Band, R.P., Rabin, H.R., Wilson, A.F., Marynowski, A., Starrevald, E., Shnitka, T.: Peripheral neuropathy related to misonidazole: Incidence and pathology. Brit. J. Cancer 37(Suppl. III): 27 l-275, 1978;